The invention relates to the use of one or more compounds chosen from compounds of the formulae Ia and Ib 
the physiologically compatible salts of the compounds of the stereoisomeric forms of the compounds of the formulae Ia and Ib, where
R1 is H or alkyl,
R2 is H, COOH, COO-alkyl or COxe2x80x94NHxe2x80x94R5,
R3 and R4 are in each case independently of one another H or OH,
n is 1, 2 or 3,
alkyl is an alkyl radical having 1 to 4 carbon atoms, and
R5 is H, alkyl, an amino acid radical, dipeptide radical or tripeptide radical
for the preparation of a cosmetic formulation
for protecting human skin from stress factors, in particular from dryness as a result of high temperatures or very low temperatures with low atmospheric humidity and/or from high salt concentration on the skin,
for protecting cells, proteins and/or biomembranes of human skin,
for protecting the microflora of human skin and/or for stabilizing the skin barrier.
Healthy human skin is colonized on its surface, the Stratum corneum, by a large number of microorganisms which live commensally. From the great diversity of these microorganisms, only a few live continually on the skin and thus form the resident skin flora. The main representatives of the resident flora on the human skin are Staphylococci, Micrococci, coryneform bacteria and Pityrospora. These live in small colonies on the surface of the Stratum corneum and in the outer epidermis. A second group of microorganisms, which settles temporarily from the outside, in particular on exposed areas of skin, is referred to as transient flora and cannot settle permanently on healthy skin whose microenvironment is heavily determined by the resident microflora. In various regions of the body, the composition of the skin flora varies depending on the microenvironment of the skin. The density of the microorganisms adapts to the respective skin environment so that the ecology of these regions of the body is not unbalanced by excessive settlement by microorganisms. Compared with the normal state of the skin, the number of microorganisms decreases in the case of dry skin, while the number of microorganisms increases in the case of moist skin, e.g. as a result of inflammatory changes in the case of eczema, by up to 1000-fold.
Being the barrier layer and surface of the human body, the skin is exposed to a large number of external stress factors. The human skin is an organ which, having diverse specialized cell typesxe2x80x94keratinocytes, melanocytes, Langerhans cells, Merkel cells and intercalated sensory cellsxe2x80x94protects the body against external influences. A differentiation should be made here between external physical, chemical, and biological influences on the human skin. External physical influences include thermal and mechanical influences, and the effect of radiation. External chemical influences are, in particular, the effect of toxins and allergens. External biological influences include the effect of foreign organisms and the metabolic products thereof.
The surface of the human skin is covered by a fatty film which, depending on the given ratios, is to be considered as an oil-in-water or a water-in-oil emulsion and contains numerous active ingredients, such as e.g. enzymes and vitamins, e.g. vitamin D. This fatty film, which has been formed from the lipids released from the sebaceous glands and keratinocytes, preserves the moisture of the skin and protects the body as a skin barrier against unfavorable environmental factors. This sensitive equilibrium of the skin barrier is disturbed by external or internal factors.
The microorganisms of human skin are subjected to various stress factors. For example, they can be damaged by drying out or by high salt concentrations on the surface of the skin, e.g. after perspiring, which can lead to damage of the skin barrier. However, some of these microorganismsxe2x80x94Staphylococci, Micrococci, Corynebacteria and Brevibacteriaxe2x80x94usually have the ability to form compatible solutes in order to protect against drying out or high salt concentration and thus contribute to the formation of an intact skin barrier. The compatible solutes, which are also referred to as stress protection substances, are low molecular weights of substances in cytoplasm.
Hitherto, it has, for example, been attempted to effect care or protection of human skin by hydrophilic substances which themselves bind water (E. A. Galinski, Experientia 49 (1993) 487-496). However, these hydrophilic substances bind water molecules of the water of hydration as well as free water molecules. Although this leads to a binding of water molecules, it does not lead, for example, to a protection of the hydration sheaths of cells, proteins and cell membranes.
The object was therefore to provide cosmetic formulations, the use of which overcome [sic] or at least reduce [sic] the abovementioned skin problems and in particular are [sic] suitable.
for protecting human skin against stress factors, in particular against dryness as a result of high temperatures or very low temperatures at low atmospheric humidity and/or against high salt concentration on the skin,
for protecting cells, proteins and/or biomembranes of the human skin,
for protecting the microflora of the human skin, and/or
for stabilizing the skin barrier.
Surprisingly, we have now found that this object is achieved by the use of one or more compounds chosen from compounds of the formulae Ia and Ib 
the physiologically compatible salts of the compounds of the formulae Ia and Ib, and the stereoisomeric forms of the compounds of the formulae Ia and Ib, where
R is H or alkyl,
R2 is H, COOH, COO-alkyl or COxe2x80x94NHxe2x80x94R5,
R3 and R4 are in each case independently of one another H or OH,
n is 1, 2 or 3,
alkyl is an alkyl radical having 1 to 4 carbon atoms, and
R5 is H, alkyl, an amino acid radical, dipeptide radical or tripeptide radical in cosmetic formulations.
Within the scope of the present invention, all compounds above and below chosen from the compounds of the formulae Ia and Ib, the physiologically compatible salts of the compounds of formulae Ia and Ib, and the stereoisomeric forms of the compounds of the formulae Ia and Ib are referred to as xe2x80x9cectoin or ectoin derivativesxe2x80x9d.
Ectoin-containing cosmetic formulations protect cells, protein, enzymes, vitamins, DNA, cell membranes and biomembranes of the skin against damage as a result of drying out and the withdrawal of water. The hydration effect of ectoin stabilizes the water equilibrium of the Stratum corneum and the skin barrier. Ectoin prevents dry and flaky skin.
In addition, ectoin-containing cosmetic formulations protect the microflora of the skin, which is important for an intact skin barrier, against stress as a result of drying out and high ion concentration after perspiring. The stabilization of the resident skin flora by ectoin or its derivatives is an important prerequisite for the equilibrium of the microenvironment of the skin and the formation of an intact skin barrier.
Ectoin and the ectoin derivatives are low molecular weight cyclic amino acid derivatives which can be obtained from various halophilic microorganisms. Both ectoin and hydroxyectoin have the advantage that they do not react with the cell metabolism.
DE 43 42 560 describes the use of ectoin and ectoin derivatives as moisture-donors in cosmetic products.
The compounds chosen from the compounds of the formulae Ia and Ib, the physiologically compatible salts of the compounds of the formulae Ia and Ib and the stereoisomeric forms of the compounds of the formulae Ia and Ib can be present in the cosmetic preparations as optical isomers, diastereomers, racemates, zwitterions, cations or as a mixture of these. Of the compounds chosen from the compounds of the formulae Ia and Ib, the physiological compatible salts of the compounds of the formulae Ia and Ib and the stereoisomeric forms of the compounds of the formulae Ia and Ib, preference is given to those in which R1 is H or CH3, R2 is H or COOH, R3 and R4 in each case independently of one another are H or OH and n is 2. Of the compounds chosen from the compounds of the formulae Ia and Ib, the physiologically compatible salts of the compounds of the formulae Ia and Ib and the stereoisomeric forms of the compounds of the formulae Ia and Ib, particular preference is given to the compounds (S)-1,4,5,6-tetrahydro-2-methyl-4-pyrimidecarboxylic acid (ectoin) and (S,S)-1,4,5,6-tetrahydro-5-hydroxy-2-methyl-4-pyrimidinecarboxylic acid (hydroxyectoin).
The term xe2x80x9camino acidxe2x80x9d means the stereoisomeric forms, e.g. D and L forms, the following compounds: alanine, xcex2-alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylaianine, serine, threonine, tryptophan, tyrosine, valine, xcex3-aminobutyrate, Nxcex5-acetyllysine, Nxcex4-acetylornithine, Nxcex3-acetyldiaminobutyrate and Nxcex1-acetyldiaminobutyrate. L-amino acids are preferred.
Amino acid radicals are derived from the corresponding amino acids.
The radicals of the following amino acids are preferred: alanine, xcex2-alanine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, serine, threonine, valine, xcex3-aminobutyrate, Nxcex5-acetyllysine, Nxcex4-acetylornithine, Nxcex3-acetyldiaminobutyrate and Nxcex1-acetyldiaminobutyrate.
The di- and tripeptide radicals are acid amides according to their chemical nature and decompose upon hydrolysis into 2 or 3 amino acids. The amino acids in the di- and tripeptide radicals are bonded together by amide bonds. Preferred di- and tripeptide radicals are built up from the preferred amino acids.
The alkyl groups include the methyl group CH3, the ethyl group C2H5, the propyl groups CH2CH2CH3 and CH(CH3)2 and the butyl groups CH2CH2CH2CH3, H3CCHCH2CH3, CH2CH(CH3)2 and C(CH3)3. The preferred alkyl group is the methyl group.
Preferred physiologically compatible salts of the compounds of the formulae Ia and Ib are, for example, alkali metal, alkaline earth metal or ammonium salts, such as Na, K, Mg or Ca salts, and salts derived from the organic bases triethylamine or tris(2-hydroxyethyl)amine. Further preferred physiologically compatible salts of the compounds of the formulae Ia and Ib arise by reaction with inorganic acids, such as hydrochloric acid, sulfuric acid and phosphoric acid, or with organic carboxylic or sulfonic acids, such as acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid and p-toluenesulfonic acid.
Compounds of the formulae Ia and Ib in which basic and acidic groups such as carboxyl or amino groups are present in equal number form internal salts.
The preparation of the compounds of the formula [sic] Ia and Ib is described in the literature (DE 43 42 560). (S)-1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid or (S,S)-1,4,5,6-tetrahydro-5-hydroxy-2-methyl-4-pyrimidinecarboxylic acid can also be obtained microbiologically (Severin et al., J. Gen. Microb. 138 (1992) 1629-1638).
The cosmetic formulation is prepared by converting one or more compounds chosen from the compounds of the formulae Ia and Ib, the physiologically compatible salts of the compounds of the formulae Ia and Ib and the stereoisomeric forms of the compounds of the formulae Ia and Ib optionally with auxiliaries and/or carriers into a suitable formulation form. The auxiliaries and carriers originate from the group of carriers, preservatives and other customary auxiliaries.
The cosmetic formulations based on one or more compounds chosen from the compounds of the formulae Ia and Ib, the physiologically compatible salts of the compounds of the formulae Ia and Ib and the stereoisomeric forms of the compounds of the formulae Ia and Ib are applied externally. Examples of application forms which may be mentioned are: solutions, suspensions, emulsions, pastes, ointments, gels, creams, lotions, powders, soaps, surfactant-containing cleansing preparations, oils and sprays. In addition to one or more compounds chosen from the compounds of the formulae Ia and Ib, the physiologically compatible salts of the compounds of the formulae Ia and Ib and the stereoisomeric forms of the compounds of the formulae Ia and Ib, any customary carriers, auxiliaries and optionally further active ingredients are added to the formulation.
Preferred auxiliaries originate from the group of preservatives, antioxidants, stabilizers, solubility promoters, vitamins, colorants, odor improvers.
In addition to one or more compounds chosen from the compounds of the formulae Ia and Ib, the physiologically compatible salts of the compounds of the formulae Ia and Ib and the stereoisomeric forms of the compounds of the formulae Ia and Ib, ointments, pastes, creams and gels can comprise the customary carriers, e.g. animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silica, talc and zinc oxide or mixtures of these substances.
In addition to one or more compounds chosen from the compounds of the formulae Ia and Ib, the physiologically compatible salts of the compounds of the formulae Ia and Ib and the stereoisomeric forms of the compounds of the formulae Ia and Ib, powders and sprays can comprise the customary carriers, e.g. lactose, talc, silica, aluminum hydroxide, calcium silicate and polyamide powder or mixtures of these substances. Sprays can additionally comprise the customary propellants, e.g. chlorofluorocarbons, propane/butane or dimethyl ether.
In addition to one or more compounds chosen from the compounds of the formulae Ia and Ib, the physiologically compatible salts of the compounds of the formulae Ia and Ib and the stereoisomeric forms of the compounds of the formulae Ia and Ib, solutions and emulsions can comprise the customary carriers, such as solvents, solubility promoters and emulsifiers, e.g. water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-diethylene glycol butyl ether, oils, in particular cottonseed oil, groundnut oil, wheatgerm oil, olive oil, castor oil and sesame oil, glycerol fatty acid esters, polyethylene glycols and fatty acid esters of sorbitan or mixtures of these substances.
In addition to one or more compounds chosen from the compounds of the formulae Ia and Ib, the physiologically compatible salts of the compounds of the formulae Ia and Ib and the stereoisomeric forms of the compounds of the formulae Ia and Ib, suspensions can comprise the customary carriers, such as liquid diluents, e.g. water, ethanol or propylene glycol, suspending agents, e.g. ethoxylated isostearyl alcohols, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystaline cellulose, aluminum metahydroxide, bentonite, agar agar and tragacanth or mixtures of these substances.
In addition to one or more compounds chosen from the compounds of the formulae Ia and Ib, the physiologically compatible salts of the compounds of the formulae Ia and Ib and the stereoisomeric forms of the compounds of the formulae Ia and Ib, soaps can comprise the customary carriers, such as alkali metal salts of fatty acids, salts of fatty acid half-esters, fatty acid protein hydrolyzates, isothionates [sic], lanolin, fatty alcohol, vegetable oils, plant extracts, glycerol, sugars or mixtures of these substances.
In addition to one or more compounds chosen from the compounds of the formulae Ia and Ib, the physiologically compatible salts of the compounds of the formulae Ia and Ib and the stereoisomeric forms of the compounds of the formulae Ia and Ib, surfactant-containing cleaning products can comprise the customary carrier substances, such as salts of fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinic half-esters, fatty acid protein hydrolyzates, isothionates [sic], imidazolinium derivatives, methyltaurates, sarcosinates, fatty acid amide ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable and synthetic oils, lanolin derivatives, ethoxylated glycerol fatty acid esters or mixtures of these substances.
In addition to one or more compounds chosen from the compounds of the formulae Ia and Ib, the physiologically compatible salts of the compounds of the formulae Ia and Ib and the stereoisomeric forms of the compounds of the formulae Ia and Ib, face and body oils can comprise the customary carrier substances such as synthetic oils, such as fatty acid esters, fatty alcohols, silicone oils, natural oils, such as plant oils and oily plant extracts, paraffin oils, lanolin oils or mixtures of these substances.
Further typically cosmetic application forms are also lipstick, lipcare sticks, mascara, eyeliner, eyeshadows, blusher, powder, emulsion and wax foundation, and sunscreen, presun and aftersun preparations.
The proportion of the compounds chosen from the compounds of the formulae Ia and Ib, the physiologically compatible salts of the compounds of the formulae Ia and Ib and the stereoisomeric forms of the compounds of the formulae Ia and Ib in the cosmetic formulation is preferably from 0.0001 to 50% by weight, particularly preferably from 0.001 to 10% by weight, based on the total cosmetic formulation.
Protection of the skin against drying out can be demonstrated, for example, in vivo, e.g. by known detection methods such as TEWL (tansepidermal water loss), corneometry (to determine the moisture in the skin), microtopography (to determine the roughness of the skin) or SELS (surface elevation of living skin).
Ectoin-containing formulations can, for example, protect the skin barrier against the harmful effect of sodium dodecyl sulfate (SDS). The use of a cosmetic ectoin-containing emulsion can significantly reduce the transepidermal water loss e.g. by up to 40% (FIG. 1) Skin pretreated with an ectoin-containing cosmetic formulation is insensitive toward damage of the skin barrier by the surfactant (SDS). As a result of the use of an ectoin-containing emulsion, the skin is better protected against surfactant damage of the skin and the water loss associated therewith.
An important aim of cosmetics continues to be protection of the skin against stress factors which lead to the drying out of the skin. In particular, dry air during cold or very warm weather conditions leads to a severe loss of water from the skin. Ectoin protects e.g. from a cosmetic O/W emulsion, against drying out (FIG. 2). In addition to the protection against drying out, ectoin-containing cosmetic formulations lead to skin moisture which is significantly better than a corresponding base formulation without ectoin (placebo) but which still comprises 3% glycerol. Furthermore, ectoin-containing cosmetic formulations still effect significantly higher skin moisture compared with the untreated area of skin or areas of skin treated only with the placebo even after 24 hours. Ectoin-containing cosmetic formulations protect the skin against rapid drying out, even against strongly hygroscopic silica gel which is applied directly to the skin. The moisture of the skin can be protected against drying out by the topical application of ectoin-containing cosmetic formulations over an extended period. Ectoin-containing cosmetic formulations are therefore highly suitable for prophylaxis against dry skin.
Stabilization of the biomembranes can be demonstrated e.g. in vitro. Here, use is made of the fact that propidium iodide is not taken up into the cells if the membrane of the skin cells is intact, and dead cells or cells with a damaged membrane are permeable to propidium iodide and are subject to a red coloration as a result of the absorption of propidium iodide.
By comparing cell cultures which have been pretreated with ectoin prior to damage, for example by the addition of DMS0, and cells which have not been pretreated, it is possible to ascertain, following subsequent propidium iodide treatment, the effect of the ectoin or its derivatives on the stabilization of the biomembranes.
To determine the cell membrane- and protein-damaging action of surfactants, the RBC Test can, for example, be used. For this, the erythrocytes are incubated e.g. with sodium dodecyl sulfate (SDS), for example for a period of 10 minutes. SDS destabilizes the membrane of untreated cells such that the cells are partially lysed and their contents such as the hemoglobin are released. The hemoglobin released during cell-wall damage serves as an indicator for the spectrophotometric determination of the membrane damage by SDS. By reference to the hemoglobin released, it is possible to determine the number of destroyed erythrocytes.
Ectoin protects the cells against damage by SDS (FIG. 3). The erythrocytes pretreated with ectoin are ore resistant toward membrane damage by SDS than untreated cells. The higher the ectoin concentration, the greater the protective effect against membrane damage.
The longer the cells are pretreated with ectoin, the greater the protective effect against membrane damage (FIG. 4). Stabilization of the cell membranes is both dependent on the ectoin concentration and on the duration of ectoin pretreatment. The higher the ectoin concentration and the longer the contact time on the erythrocytes, the greater the cell membranes are protected.
Stabilization of the resident microflora can, for example, be demonstrated in vivo. Following ectoin treatment of certain areas of skin, for example the forearms, the skin is subjected e.g. to dry and/or heat stress in a climatically controlled chamber. The bacteria from the forearms are then isolated, and a xe2x80x9cliving cell count determinationxe2x80x9d is carried out using vital staining and a growth curve for determining the kinetics, for example by plating out the bacteria on culture plates (plate method) or by the impedance method using conductivity measurements. A comparison of these results with those for areas of skin not pretreated provides evidence of the effect of ectoin or its derivatives on the stabilization of the resident microflora.
All compounds or components which can be used in cosmetic formulations are either known and available commercially or can be synthesized by known methods.
The examples below serve to illustrate the invention and are in no way to be regarded as a limitation. All percentages are percentages by weight.
The INCI names of raw materials used are as follows (the INCI names are by definition given in English):